10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr.

Chair

Prof.

Elucidating the biotransformation of the heat induced contaminant 2,5-dimethylfuran

General Toxicology

TOX08

Furan and methylfurans, including 2,5‑dimethylfuran (DMF) are formed as heat induced contaminants in a variety of thermally processed food products. It is generally assumed that their reported hepatotoxicity and potential carcinogenicity appears to be primarily attributed to cytochrome P450 (CYP) mediated epoxidation leading to the formation of reactive α,β‑unsaturated dicarbonyls which are capable of forming adducts with various cellular nucleophiles. While an analogue biotransformation is assumed for DMF, resulting in the formation of cis‑3‑hexene‑2,5‑dione (HDO), unilateral side-chain hydroxylation represents an alternative metabolic pathway for DMF. It is hypothesized that the potentially formed 5‑methyl‑2‑furfuryl alcohol (MFA) is subsequently oxidized further to 5‑methyl‑2‑furfural (MFF) and 5‑methyl‑2‑furancarboxylic acid (MFC). Therefore, side‑chain hydroxylation is expected to represent the major detoxification pathway for DMF.To investigate potential phase I metabolites of DMF in human liver microsomes, a HPLC‑MS/MS method was established to quantify formed HDO after scavenging with glutathione. In parallel, formation of the hydroxylated metabolite MFA as well as further oxidized metabolites MFF and MFC were monitored by GC‑MS. Thus, comparison of HDO and MFA formation kinetics in human liver microsomes was possible. In addition, our experimental approach was used with selected isoenzyme systems in order to identify the CYP isoforms involved in the biotransformation of DMF.We were able to show that metabolic activation of DMF to HDO represents the primary biotransformation pathway of DMF. In contrast, the parallel detoxification via side-chain hydroxylation proceeds significantly slower in the investigated systems. Furthermore, our findings indicate significant differences in the metabolism of DMF compared to structurally related furan analogues such as 2‑methylfuran. Overall, this work provides more detailed insights into the biotransformation of methylfurans as well as the molecular mechanisms of their toxicity and contributes to an improved risk assessment of these heat induced contaminants.Funding: DFG RI 1176/13-1

Toxicology

Short talk

Biomonitoring of heat-induced food contaminants: In vivo metabolism and excretion kinetics of alkylated furans to define biomarkers of exposure

Heat-processing of food gives rise to a variety of process-related food contaminants, including furan and alkylated furans such as 2-methylfuran (2-MF), 2-ethylfuran (2-EF), and 2-pentylfuran (2-PF). Based on the available data, it appears that alkylfurans may significantly add to furan exposure and thus further increase health concerns related to the presence of furan(s) in food. However, due to limited data on alkylated furans in food and variations in dietary patterns, current exposure assessment may be unreliable. A biomarker-based approach may enable more accurate exposure estimates, yet there are currently limited data on the in vivo biotransformation and toxicokinetics of alkylated furans as a basis for identifying and validating potential biomarker of exposure.The aim of the present study was to investigate the in vivo biotransformation and toxicokinetics of alkylated furans by identifying and monitoring urinary metabolites of 2-MF, 2-EF and 2-PF in F344 rats exposed to a single oral dose of 1,000 µg alkylated furan / kg body weight via LC-MS/MS. It is assumed that - in analogy to furan - alkylfurans may undergo cytochrome P450 mediated epoxidation of the double bond, giving rise to alkyl substituted dialdehydes similar to the furan metabolite cis-2-butene-1,4-dial. In line with this, we identified metabolites of 2-MF, 2-EF and 2-PF that derive from the reactive intermediates (cis-2-oxopent-2-enal, cis-4-oxohex-2-enal and cis-4-oxonon-2-enal, respectively) and subsequent conjugation with glutathione (GSH) and amino acids, such as lysine and cysteine. Similar to our previous observations with furan, the N-acetyllysine adducts showed substantial endogenous background levels, whereas GSH-dependent metabolites of 2-MF, 2-EF and 2-PF were without background occurrence and were rapidly excreted within the first 24h post-exposure. Of note, the overall excretion rate of GSH and amino acid-derived adducts of the alkylfurans was significantly lower compared to furan, which may indicate reduced bioactivation of alkylated furans as compared to furan. Overall, results from this study suggest that GSH-adducts of alkylated furans may be suitable as urinary biomarkers of exposure, although sensitive detection of these metabolites at doses relevant to human exposure may be challenging.

How does the lipophilicity and volatility of monoterpenes affect their toxicity in vitro?

Interpretation of in vitro toxicity tests is usually based on calculated nominal concentrations (cnom) while the concentration that is bioavailable to the test system (cfree) may be significantly lower. Depending on the physicochemical properties of the test substance and the assay setup, this discrepancy can occur e.g. due to volatilization, sorption to culture vessel material or serum constituents. If not accounted for, different routes of test substance depletion may reduce the sensitivity of the in vitro test, leading to possible underestimation of toxicity or even false negative results1.According to the OECD, test substances with a log KOW>4 are prone to sorption and volatilization can be expected for test substances with a Henry volatility of Hvpc>0.1 Pa m3 mol-1. For these 'difficult test chemicals', analytical confirmation of the exposure concentration as well as modifications of the assay setup are recommended to account for and minimize concentration depletion2.For the widely used monoterpenes (R)-limonene (RLIM) and β-myrcene (βMYR) and the more polar monoterpenoid linalool (LIN) low toxicity is reported3–5. However, regarding the physicochemical properties of RLIM and βMYR significant concentration depletion was expected, which has not been considered in previous studies6–7.Therefore, we assessed the mutagenicity and cytotoxicity of RLIM, βMYR and LIN towards Salmonella Typhimurium via resazurin reduction assay and Ames fluctuation test, respectively and genotoxicity via the mouse lymphoma assay. A polystyrene-free incubation method was included to prevent sorption of the test substance and a headspace-free (hs-free) incubation method to minimize volatilization. Concentration analysis via headspace gas chromatography-mass spectrometry (hs-GC-MS) revealed, that the monoterpenes evaporated quantitatively during the first minutes of conventional incubation. On the other hand, hs-free incubation enabled sufficient exposure over the entire incubation period and resulted in lower nominal EC50-values compared to the other setups. No signs for mutagenicity or genotoxicity were observed in all incubation setups.1Groothuis et al., Toxicology 2015, 332; 2OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2019; 3IARC, IARC Monog Eval Carc 1999, 73; 4IARC, IARC Monog Eval Carc 2019, 119; 5Letizia et al., Food Chem Toxicol, 2003 41 (7); 6https://pubchem.ncbi.nlm.nih.gov; 7Sander, Atmos Chem Phys, 2023 23 (19)

Short talk 3
Food Toxicology

Appointment

Chair

Programme

Short talk 3Food Toxicology

Appointment

Chair

Programme

Short talk 3
Food Toxicology